The analysis of biological and other compounds often begins with the separation of macromolecules. Capillary electrophoresis has proved a valuable tool for the separation of macromolecules, allowing their further analysis or isolation. Compounds may be separated by size, charge, affinity to complexing agents etc.
Capillary electrophoresis systems present various advantages. First, the low sample volume consumption required for capillary electrophoretic separation reduces costs for sample analysis. Second, the high surface to volume ratio, characteristic of a capillary dimension microchannels, allows for efficient heat dissipation. The resulting lower joule heating of the capillary allows greater voltage to be used in separations. Using higher voltages makes the separation more rapid, mitigating against diffusion from bandspreading. Third, capillary electrophoresis systems are readily adaptable to high throughput sample processing. With rapid increase in genomic information, the need for the rapid analysis of nucleic and samples is increasingly acute. Capillary electrophoresis systems are readily adaptable to multiplexing to increase sample throughput. Capillary dimension microchannels may be designed as an array of microchannels, such as a group of capillary tubes in a capillary array electrophoresis system (CAE system) or as a plurality of lanes on an electrophoresis chip. In addition, the use of multiple detectable labels that may be separately detected by a detector allow a second level of multiplexing, further increasing throughput. Finally, various configurations of sample pooling allow another level of multiplexing.
Capillary electrophoresis requires a microchannel (generally of capillary tube dimensions) that is filled with a separation media. The sample is introduced at one end of the microchannel and after migration through the length of the channel is detected at the opposite end. The separation media contains an electrolyte buffer allowing the current to flow from an anode to a cathode. In one common method of capillary electrophoresis, a polymer included in the separation medium acts as a sieve for the separation of biomolecules. The movement of the biomolecules is inhibited by the sieving matrix, with smaller molecules able to overcome the friction and to move more quickly through the matrix than larger molecules.
U.S. Pat. No. 4,865,706 discloses the use of crosslinked polyacrylamide gel as a molecular sieving agent for the separation of charged molecules. The interior wall of the capillary is covalently modified with a bonding agent. Typically, polymerization of a crosslinked separation matrix takes place inside the capillary tube. The mixture of monomer and a cross-linker is injected into the capillary and catalytically converted to a polymerized form. This may result in incomplete polymerization formation of bubbles and lack of gel uniformity, issues which represent the main drawback of the cross-linked separation matrices. Another drawback is the difficulty in removing the crosslinked gel.
U.S. Pat. Nos. 5,089,111; 5,374,527 and 5,370,777 describe a significant improvement in CE matrix technology by replaceable matrix composed of an entangled solution of linear polymer. Although less rigid than crosslinked gel, the network of dynamic pores in entangled matrix provides equivalent media for separation of biomolecules. The main advantage of the entangled polymer media is the preparation outside the capillary resulting in a high homogeneity of the matrix. Another significant advantage is the ability to replace the matrix in capillaries after each run using a high pressure, thus increasing the run to run reproducibility.
Various polymers have been proposed as entangled media for the separation of charged biomolecules. U.S. Pat. No. 5,567,292 proposes the use of a polymer for both suppression of electroosmotic flow and as a matrix for separation of polynucleotides in the size gauge of about 100 to 500 nucleotides in an uncoated capillary. The separation compound would be water-soluble, lack charged groups in a liquid media of 6-9 pH, have a molecular weight of 5×103 to 1×106 daltons and concentration between 0.001 and 10%. These separation media include polylactams, such as polyvinylpyrrolidone, as well as substituted polyacrylamide derivatives.
With these types of media entanglement is a prerequisite for an efficient separation of oligonucleotides in the gauge from approximately 100 to 1000 bases in length.
The viscosity of an entangled medium requires high-pressure introduction into the capillary. In addition, newly developed CE methods utilizing multiple electrokinetic injections of samples are not compatible with classic entangled matrices. The multiple sample loads as well as interruption in the current required in multiple injections is believed to degrade the entangled matrix. A primary feature of an entangled matrix is the high sieving capacity attendant with the relatively high molecular weight of the sieving polymers. However, this feature is not required for the separation of short oligonucleotides. Assays for SNP typing; such as single-nucleotide primer extension (SnuPE) or oligonucleotide ligase assay (OLA); produce short oligonucleotide fragments (100 bases or smaller). To avoid the requirement of high pressure fill and matrix inhomogeneity due to the sample impact, an alternative separation media that provides high separation speed and resolution of small oligonucleotide fragments is desired.
In 1993, a modification of free-zone CE used for separation of small ionic compounds was developed. In this method, dilute linear polymers are added to the CE buffer electrolyte. These polymeric additives act as pseudo-phases for separation. In contrast to sieving, pseudo-phase leads to separation of sample mixture through hydrogen bonding, hydrophobic interactions, steric interactions, and dipole-dipole interactions. However, to date the use of these polymers for pseudo-phase separation has been fairly limited. In one example a high-molecular weight polyvinylpyrrolidone [PVP] was used at low concentration as a buffer additive for the capillary electrophoresis separation of diastereomeric derivatives of tryptophan. Schutzner, et al., J. Chromatogr. 639 (1993) 375-378. In a similar manner, a combination of polyethylene glycol and polyvinylpyrrolidone were used for the electrophoretic separation of azo dye compounds based on their inherent hydrophobicity. Blatny et al., J. Chromatogr. 717 (1995) 157-166. Similar high-molecular PVP matrix was applied for separation of nucleotide adducts (Barry et al.). These studies provided an initial indication that PVP could be used as a pseudophase for the separation of hydrophobically modified oligomers.
One of the most frequently used detection techniques in CE is laser-induced fluorescence. Because DNA molecules exhibit no native fluorescence, synthetic dyes are commonly introduced at the 5′-end of each DNA molecule. Most fluorescent dyes include aromatic rings, capable of excitation of PI-electrons and subsequent emission of fluorescence light. By nature, the aromatic rings exhibit inherent hydrophobicity, which can be directly utilized for interaction with PVP pseudophase matrix leading to chromatography-like separation.
It is an object of the invention to provide a separation matrix and attendant method for using the separation matrix, which is able to provide high resolution of short fluorescently labeled oligonucleotide fragments (10-100 base fragments).
It is a further object to provide a lower viscosity separation media. Such a media could be introduced into a capillary with low-pressure nitrogen filling.
It is a further object of the invention to provide a separation media that does not require a complex polymerization and does not exhibit entanglement property such as sieving. It is a further object to provide a separation procedure for short oligonucleotide fragments that shows superior peak spacing (indicating separation selectivity) and peak efficiency.
It is a further object to allow the use of higher voltages than are allowed with the use of a separation media containing an entangled polymer matrix.
It is a further object to provide a CE media that may be used for a multiple injection procedure for separation samples containing small compounds.